Hanseniaspora uvarum prolongs shelf life of strawberry via volatile production

Food Microbiology 63 (2017) 205e212

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Hanseniaspora uvarum prolongs shelf life of strawberry via volatile production Xiaojie Qin a, b, 1, Hongmei Xiao a, *, 1, Xu Cheng c, Hailian Zhou d, Linyuan Si a a

Key Laboratory of Quality and Safety Risk Assessment in Agricultural Products Preservation (Nanjing), Ministry of Agriculture /College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu, 210095, PR China b MOST-USDA Joint Research Center for Food Safety and Bor Luh Food Safety Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, 200240, PR China c Laboratory of Molecular Biology, Department of Plant Sciences, Wageningen University, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands d Centre Testing International, Shanghai, 201206, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 April 2016 Received in revised form 1 November 2016 Accepted 6 November 2016 Available online 9 November 2016

Gray mold caused by Botrytis cinerea led to severe postharvest losses for strawberry industry. In recent years, some studies have shown that postharvest diseases of strawberry can be controlled by using bacterial, fungal and yeast strains. The yeast strain Hanseniaspora uvarum was shown as an effective antagonist against B. cinerea growth. Here, we further investigated the volatile organic compounds (VOCs) production of H. uvarum and how this could impact on postharvest gray mold control of strawberry. A total of 28 VOCs were detected by GC-MS in the headspace of H. uvarum and strawberry with/without B. cinerea (SI and RSI
800). Among these VOCs, 15 VOCs were detected in both conditions, 4 VOCs were H. uvarum and strawberry without B. cinerea and the other 9 VOCs were only detected when B. cinerea was inoculated. Two VOCs, ethyl acetate and 1,3,5,7-cyclooctatetraene, enhanced by inoculation of B. cinerea. In in vitro assay, H. uvarum significantly inhibited mycelial growth and spore germination of B. cinerea via VOCs production. Moreover, in vivo assay showed that H. uvarum reduced B. cinerea infection of strawberry and maintained fruit appearance, firmness and total soluble solids via VOCs production. Collectively, our results showed that H. uvarum VOCs significantly controlled postharvest gray mold of strawberry and prolonged the storage time and shelf life. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Volatile organic compounds Antagonistic yeast Hanseniaspora uvarum Botrytis cinerea Bio-fumigation Strawberry

1. Introduction Strawberries (Fragaria ananassa Duch) are highly perishable fruits due to their extreme tenderness, vulnerability to mechanical damage, high level of respiration and their susceptibility to fungal spoilage (Dennis, 1978). Among them, gray mold caused by Botrytis cinerea is responsible for severe preharvest and postharvest losses for strawberry industry (Romanazzi et al., 2001; Droby and Lichter, 2004). B. cinerea is a necrotrophic plant pathogen. It can secrete cell-wall-degrading enzymes such as cellulases, hemicellulases, polygalacturonases, pectin methylesterases, and phytotoxic metabolites such as botrydial, which facilitate infection of strawberry fruit and developments of soft rot symptoms. Thus far, there are no strawberry cultivars with high resistance to B. cinerea (Huang et al.,

* Corresponding author. E-mail address: [email protected] (H. Xiao). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.fm.2016.11.005 0740-0020/© 2016 Elsevier Ltd. All rights reserved.

2011). More efforts have been made to minimize losses through developing a better understanding of mechanisms of postharvest diseases, as well as by developing adequate postharvest handing technologies and control strategies (Prusky and Gullino, 2010). In recent years, biological control is becoming an increasingly effective measure to control postharvest diseases of fruit (ElNeshawy and Shetaia, 2003; Zhang et al., 2007; Fan et al., 2009; Costa et al., 2013; Maryam et al., 2014; Zhang et al., 2014; Parafati et al., 2015; Platania et al., 2012). The mechanisms of biocontrol agents interact with pathogens and fruit tissues including competition for limiting nutrients and space, induced resistance, produced of lytic enzymes, mycoparasitism and the role of oxidative stress are demonstrated (Spadaro and Droby, 2016). Biocontrol applications for postharvest disease control is now directed more towards the use of volatile organic compounds (VOCs) produced by microorganisms that are biodegradable, that do not leave toxic residues on the fruit surface (Mercier and Smilanick, 2005). Biological fumigation, or biofumigation, with volatile

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compounds produced by microorganisms has shown promise for killing a wide range of storage pathogens and controlling fungal decay. Mercier and Smilanick (2005) indicated that VOCs produced by Muscador albus had possibility to control fungal decays of apple and peach by biofumigation. Li et al. (2012, 2010) found that volatile substances from Streptomyces globisporus JK-1 inhibited the spore germination and mycelial growth of Botrytis cinerea and Penicillium italicum on tomato and Citrus microcarpa, respectively. Zheng et al. (2013) and Chen et al. (2008) indicated the VOCs of Bacillus spp. were antagonistic to the Botrytis cinerea, Colletotrichum gloeosporioides, Penicillium digitatum, Penicillium italicum and Penicillium crusto sum. Garbeva et al., 2014 analyzed the composition of volatiles produced by Collimonas on agar under different nutrient conditions and studied the effect on fungal growth. The volatiles had a negative effect on the growth of a broad spectrum of fungal species. Antifungal volatiles produced by Collimonas played an important role in realizing its mycophagous lifestyle. Di Francesco et al. (2015) demonstrated that the conidia germination of Penicillium spp. was completely inhibited by VOCs produced by two Aurebasidium pullulans L1 and L8 strains (100% inhibition). Moreover, the production of VOCs could play an essential role in the antagonistic activity of two A. pullulans strains against five fruit postharvest pathogens in vitro and in vivo tests. In our previous research, Hanseniaspora uvarum was an effective antagonist, which reduced the natural decay development of grape and strawberry and maintained the quality parameters (Qin et al., 2015; Cai et al., 2015). Meanwhile, H. uvarum has been reported as a potential biological control agent for control of chilli fruit rot (Basha and Ramanujam., 2015) and postharvest green mold of oranges (Li et al., 2016). H. uvarum has been reported to inhibit the growth of B. cinerea with multiple modes of action such as competition for nutrients and space, induction of host defense, morphology change and secondary metabolites (Liu et al., 2010; Qin et al., 2015; Cai et al., 2015; Romanazzi et al., 2012). Moreira et al. (2011) have identified different VOCs produced by Hanseniaspora yeasts during red wine vinifications included such as 3methyl-1-butanol, ethyl acetate, phenylethyl alcohol and butanoic acid, ethyl ester. However, the effect of VOCs produced by H. uvarum as a mode of antagonism action for the control of postharvest disease is not described. Here, we identified VOCs from H. uvarum on strawberry with/without B. cinerea, and studied antifungal activity of VOCs against B. cinerea in vitro and in vivo. This work attempted to find a better strategy to control gray mold of strawberry, and prolong the storage time and shelf time. 2. Materials and methods 2.1. Fungal strains The antagonist yeast was isolated from the surface of strawberries and identified as Hanseniaspora uvarum based on the similarity analysis of its morphologies, physiological-biochemical characteristics and 26S rDNA D1/D2 domain sequence (Genbank accession number: JX125041). The yeast was maintained on potato dextrose agar (PDA, 200 mL extract of boiled potatoes, 20 g dextrose and 20 g agar in 1000 mL distilled water) at 4  C before use. Liquid cultures of the yeast were grown in 250 mL Erlenmeyer flasks with 100 mL potato dextrose (PDB, 200 mL extract of boiled potatoes, 20 g dextrose in 1000 mL distilled water) on a gyratory shaker at 180 r min1, 28  C for 24 h. The yeast cells were acquired by centrifuging at 6000 g for 15 min at 4  C, then washed with sterile distilled water twice and re-suspended in sterile distilled water with 0.05% Tween-20. Cells concentration was adjusted to a final concentration of approximately 1  109 CFU mL1 using a hemacytometer (XB-K-25; Shanghai, China).

B.cinerea, from infected strawberry fruit, obtained from Kang Tu, Department of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China (Wei et al., 2014) and stored on potato dextrose agar (PDA) at 4  C. Before use, B. cinerea was freshly cultured on PDA plates at 23  C. Spore suspensions were prepared by removing the spores from a 7-day old culture with a sterile inoculator and then suspending in sterile distilled water to the required concentration of 1  105 spores$mL1, which was estimated using a hemacytometer. 2.2. Fruit Fragaria ananassa ‘Hong Yan’, were grown in a greenhouse located in Yuhua district, Nanjing city, Jiangsu province, China. Diurnal temperature in the greenhouses ranges from 8  C to 25  C. Commercially mature fruit were harvested early in the morning, and then transported immediately to the laboratory in 2 h. All fruits were selected depending on the maturity, size color, and the absence of physical injuries or infections. 2.3. Gas chromatography-mass spectrometry analysis of H. uvarum VOCs The equator of fruits were hit a wide 3 mm, deep 4 mm hole with a sterile hole puncher and placed 30 min. The experiments were divided into three groups: (Control) Inoculated with 100 mL sterile water into the wound of strawberry fruit; (H) Inoculated with 100 mL 1  109 CFU mL1 H. uvarum into the wound of strawberry fruit; (HB) Inoculated with 100 mL 1  109 CFU mL1 H. uvarum into the wound of strawberry fruit and then 100 mL 1  105 spore$mL1 conidial suspension of B. cinerea. All groups were incubated for 3 days at 25  C and 5 g wound samples of every group were transferred to a 20 mL extraction glass vial. The experiment was repeated twice per group (15 unwounded fruits/ group). Yeast VOCs composition was evaluated by SPME coupled with gas chromatography-mass spectrometry (GC-MS). For all subsequent experiments, an SPME fiber with 75 mm CAR/PDMS coating was used. Trapped compounds were desorbed into the GC injection port at 250  C for 3 min, and separated in a gas chromatograph equipped with a TR-5MS fused silica capillary column (30 m by 0.25 mm inside diameter; 0.25 mm film thickness) connected to a quadrupole mass detector. The oven temperature was set at 40  C for 2.5 min and then programmed to rise from 40 to 200  C at 5  C$min1, from 200 to 240  C at 10  C min1 for 5 min. The transfer line was heated at 230  C and the ion source at 250  C. Helium carrier gas had a flow of 1 mL min1. The mass spectrometer was operated in electron impact mode at 70 eV, scanning the range of 40e400 amu. Identification of the VOCs was done by comparing the mass spectra and retention times of the individual VOC with those for the standard compounds deposited in the database of the National Institute of Standards and Technology (NIST) and the Wiley Registry of Mass Spectral Database (Wiley 7.0) in the MS (SI and RSI
800). 2.4. In vitro VOCs antagonistic assay The efficacy of the VOCs produced by the yeast on the mycelium growth and conidia germination of B. cinerea were tested. The method used was adopted by a double petri dish assay (Rouissi et al., 2013) with some modifications. The PDA plates were inoculated spreading 100 mL of antagonist cell suspension (1  109 CFU mL1). Equivalent amounts of sterile distilled water were used as the control. The lid of plate was replaced, by a base plate of PDA inoculated with an agar plug (5 mm) from the

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periphery of an actively growing culture of B. cinerea. The two base plates were sealed immediately with double layer of parafilm and incubated for 5 days at 25  C. The fungal hyphal diameter was assessed and the percentage of inhibition was calculated using the formula: (Control value  Treatment value)/Control value  100%. Spore suspensions (1  105 spore$mL1) were prepared from exposed to volatiles treatment and control on the fifth day and then inoculated in PDB. Four hours later, conidial germination and germ tube length were examined via optical light microscope. For each experiment, there were three replicates, and the experiments were repeated twice. 2.5. In vivo VOCs antagonistic assay 2.5.1. Effects of H. uvarum VOCs on control of gray mold in strawberry fruit at 25  C The bioassay was done in closed glass desiccators (6.5 cm by 15 cm, down diameter by up diameter). Aliquots of the yeast cell suspension of H. uvarum (1  109 CFU mL1) were pipetted and plated on PDA in petri dishes at 200 mL of yeast cell suspension per dish. Thirty unwounded strawberry fruits of similar size individually were inoculated with the conidial suspension (z1  105 spore$mL1) of B. cinerea on the surface of strawberry fruit for each treatment. The dishes with the culture of H. uvarum were placed at the bottom of the desiccators at six dishes per desiccator. The fruit were then placed on the perforated ceramic clapboard above the uncovered dishes containing H. uvarum cultures or uncolonized PDA in a desiccator. There were four different fumigation time treatments in each trial: (i) negative control (no fumigation), (ii) one day, (iii) two days, (iv) three days. After different fumigation time, strawberries were placed in an incubator at 25  C (RH90%e 95%). The decay index and weight loss were evaluated on the 6th day. The experiment was repeated twice with three replicates per treatment (30 fruits/treatment). An additional experiment was conducted to verify the suppressive effect of the VOCs of H. uvarum on development of gray mold. Thirty unwounded fruits were inoculated with the conidial suspension (z1  105 spore$mL1) of B. cinerea on the surface of strawberry fruit for each treatment. There were three treatments in this experiment: (i) uninoculated with the yeast suspension on PDA, (ii) inoculated with the yeast suspension on PDA, (iii) inoculated with the yeast suspension on PDA and active carbon. There were three desiccators (replicates) for each treatment. For the (i) treatment, six uncovered dishes with uninoculated PDA were placed at the bottom of each desiccator. For the (ii) treatment, six uncovered dishes with culture of H. uvarum were placed at the bottom of each desiccator. For the (iii) treatment, six uncovered dishes with the PDA cultures of H. uvarum and 50 g of active carbon were placed at the bottom of each desiccator. The desiccators were individually covered and incubated at 25  C (RH90%e95%) for 6 days. The decay index and weight loss were determined after incubation. The experiment was repeated twice with three replicates per treatment (30 fruits/treatment). 2.5.2. Effects of VOCs on postharvest quality of strawberry fruit during cold storage The bioassay was done in closed glass desiccators (9 cm by 27 cm, down diameter by up diameter). Aliquots of the yeast cell suspension of H. uvarum (1  109 CFU mL1) were pipetted and plated on PDA in petri dishes at 500 mL of yeast cell suspension per dish. Control: no inoculated yeast suspension on PDA; Treatment: inoculated yeast suspensions on PDA. Seventy five of unwounded strawberry fruits were then placed on the perforated ceramic clapboard above the eight uncovered dishes containing H. uvarum

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cultures or uncolonized PDA in a desiccator for each treatment. The desiccators were individually covered and incubated at 25  C (RH90%e95%) for 3 days, Then all groups were taken out from the desiccators and placed at 2 ± 1  C, RH90%e95%. Postharvest qualities of the fruit were evaluated at intervals of 5 days. Weight loss was determined before treatment and after storage, and the weight loss was calculated as (A-B)/A  100%, A: weight before treatment, B: weight after storage. Commodity rate was calculated as (A þ B)/ C  100%, where A was the number of no decay, B was the number of very slight decay that covering <10% of the fruit surface and C was the total number of fruit. The testing methods of decay index, firmness, pH and total soluble solids (TSS) were same with Cai et al., 2015. The experiment was repeated twice with three replicates per treatment (75 fruits/treatment). 2.6. Statistical analysis All statistical analyses were performed in the SAS Software (Version8.2; SAS Institute, Cary, NC, USA). The data were analyzed by one-way analysis of variance (ANOVA). Comparison of means was performed by Duncan’s multiple range tests. Statistical significance was assessed at the level of P  0.05. 3. Results 3.1. Identification of VOCs from H.uvarum on strawberry with/ without B.cinerea A total of 28 volatile organic compounds were detected from H. uvarum on strawberry with/without B. cinerea, excluding the VOCs of strawberry (SI and RSI
800) (Table 1). Among these VOCs, 15 VOCs were detected in both conditions, 4 VOCs were H. uvarum and strawberry without B. cinerea and the other 9 VOCs were only detected when B. cinerea was inoculated. These volatiles were classified into alcohols, esters, organic acids, alkenes, ketones and aldehydes (Table 1). In the presence of B. cinerea, the relative peak areas of ethyl acetate and 1,3,5,7-cyclooctatetraene was higher than that of H. uvarum alone, approximately 1.6- and 17.4-fold (Table 1 and Fig. 1A&B). Through analyzing the composition of H. uvarum VOCs, we detected several VOCs which have been demonstrated inhibiting fungal growth (specific annotation in Table 1). Therefore, we tested the antifungal activity of H. uvarum via VOCs production in vitro and in vivo. 3.2. Effect of H.uvarum VOCs against B.cinerea Growth of B. cinerea was monitored and mycelial length was quantified daily for 5 days. The in vitro assay showed that H. uvarum was able to inhibit growth of B. cinerea via production of VOCs (Fig. 2A). A significant difference (P < 0.05) in the fungal hyphal diameter of B. cinerea was observed between the H. uvarum treatment and the control (Fig. 2B). H. uvarum affected B. cinerea development via VOCs production as well. In control B. cinerea started sporulating 3 days after inoculation, however, sporulation was not observed in the H. uvarum treatment (Fig. 2A). Futhermore, microscopy analysis revealed differences in hypha morphology of B. cinerea between H. uvarum treatment and control. The hypha of B. cinerea in control showed a thick morphology with a disperse surface. However, the stunted tips and morphological abnormalities on hypha of B. cinerea were observed in the H. uvarum treatment (Fig. 2C). These results suggested that the VOCs had a significant inhibitory effect on B. cinerea growth.

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3.3. Effects of H. uvarum VOCs on control of gray mold in strawberry fruit at 25  C After incubation at 25  C, RH90%e95% for 6 days, all strawberry fruits in the control treatment (uncolonized PDA) showed soft rot and gray mold symptoms, with the highest decay index and weight loss (Fig. 3). However, in the presence of the fumigation of H. uvarum VOCs increasing from 1st day to 3rd day, the decay index of strawberry decreased from 0.71 to 0.29 and the weight loss decreased from 2.45 to 0.73, which were significantly lower than those of the control fruit (P < 0.05) (Fig. 3). The H. uvarum VOCs suppressed significantly decay index and weight loss of strawberry (Fig. 4). In contrast, the suppressive effect of the H. uvarum VOCs and active carbon treatment was greatly nullified by active carbon. The decay index reached 0.59 (same with the control) and weight loss reached 4.99% (significantly higher than the control) in this treatment. These values were significantly higher than those in the H. uvarum treatment (P < 0.05) (Fig. 4). 3.4. Effects of H. uvarum VOCs on postharvest quality of strawberry fruit during cold storage The effects of H. uvarum via VOCs on strawberry fruit decay index, commodity index, weight loss, firmness, total soluble solid and pH were determined (Fig. 5). Results showed that the decay index of strawberry treated by the H. uvarum treatment was 0.65, which was significantly (P < 0.05) lower than that of the control (Fig. 5A) on the 25th day. Meanwhile, the commodity rate of strawberries treated by H. uvarum treatment was 33.3%, which was

Fig. 1. (A) Total ion chromatogram of VOCs produced by H. uvarum on strawberry. (B) Total ion chromatogram of VOCs produced by H. uvarum on strawberry inoculated with B. cinerea. (C) The structures of ethyl acetate and 1,3,5,7-cyclooctatetraene.

significantly (P < 0.05) higher than that of the control (0%) on the 25th day (Fig. 5B). The weight loss of fruit treated by the H. uvarum

Table 1 Volatile organic compounds detected from H.uvarum on strawberry with/without B.cinerea. Possible compounds

RA (%)a H. uvarum

H. uvarum and B. cinerea

Ethanol Ethyl acetate Propanoic acid, ethyl ester 3-Methyl-1-butanol 1,3,5,7-Cyclooctatetraene Phenylethyl alcohol Acetic acid, 2-phenylethyl ester Butanoic acid, 2-methyl-, ethyl ester 1-Hexanol Butanoic acid 3-hydroxy-, ethyl ester Benzyl alcohol Hexanoic acid, 3-hydroxy-, ethyl ester Octanoic acid, ethyl ester Benzenepropanol n-Decanoic acid 1-Octanol Acetophenone Acetic acid, octyl ester Hexanoic acid, 2-hexenyl ester, (E)2-Nonanone 3-Methyl-1-butanol, acetate Phenol, 4-ethylAcetic acid 2-methylpropyl ester Hexanoic acid 3-Phenyl-2-propenal Decanoic acid, ethyl ester 3-Phenyl-2-propenoic acid, ethyl ester Dodecanoic acid

2.01 21.78 1.06 3.19 1.27 2.14 1.41 0.19 0.81 0.81 0.12 0.09 0.76 0.27 0.06 0.25 0.08 0.07 0.06 e e e e e e e e e

1.83 34.87 0.98 1.86 22.1 1.55 1.14 0.12 0.32 0.54 0.16 0.12 0.57 0.17 0.17 e e e e 0.11 0.03 0.17 0.21 0.41 0.03 0.05 0.93 0.14

SI/RSIb

MW(Da)c

Antifungal activity

References

892/892 900/901 832/891 895/892 898/908 944/946 923/923 867/908 894/906 926/927 915/917 909/914 924/925 807/901 909/913 906/911 888/888 901/904 897/973 807/879 882/907 840/860 820/820 800/800 839/878 914/916 907/912 801/801

46 88 102 88 104 122 164 130 102 132 108 160 172 136 172 130 120 172 198 142 130 122 116 116 132 200 176 200

✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ※ ※ ※ ※ ※ ※ ※ ※ ※ ※ ※ ✓ ✓ ✓ ※ ※ ※ ※ ※ ※

Huang Huang Huang Huang Huang Huang Huang Huang

et et et et et et et et

al., al., al., al., al., al., al., al.,

2011 2011 2011 2011; Singh et al., 2011 2011 2011 2011 2011; Kudalkar et al., 2012

Huang et al., 2011 Huang et al., 2011; Zheng et al., 2013 Ren et al., 2010

e, Not detected or not in the scope (SI and RSI
800). ✓, These volatiles have been demonstrated the antifungal activity in other reference according to the above table. ※, These volatiles were detected in this study but not sure the antifungal activity. a RA, relative peak area: the value for a volatile compound represented the percentage of the area of the peak for that volatile compound in the total area of peaks for all the detected volatile compounds. b SI, Similarity index; RSI, Reverse similarity index; the chemical compounds (SI and RSI
800) were analyzed. c MW, molecular weight.

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C

Control

H.uvarum

Fig. 2. (A) Fungal growth after exposure to H. uvarum VOCs. Photographs of all the plates were taken with a fixed distance between the plate and the camera. (B) In vitro antifungal activity with H. uvarum VOCs. Bars represent standard errors of the mean of 3 independent replicates. Asterisks indicate statistical differences compared to control according to Duncan’s multiple range test at P  0.05 level. (C) Microscopic view of B. cinerea hyphae exposed to H. uvarum VOCs on the 5th day (40  ).

treatment was lower than that of control significantly (P < 0.05) (Fig. 5C). Besides, the treatment of H. uvarum maintained greatly the fruit firmness, total soluble solid and pH of strawberry (Fig. 5DeF), which were significantly (P < 0.05) higher than that of control on the 25th day. 4. Discussion In previous research, H. uvarum played an important role in biocontrol of gray mold of strawberry by nutrient and space competition, suppression of conidial germination and hyphal growth of B. cinerea, hyperparasitism and induction of host defense (Qin et al., 2015; Cai et al., 2015). However, the antagonistic mechanisms of H. uvarum were not fully understood. In this study we focused on antifungal activity of the secondary metabolites, the volatile organic compounds (VOCs) produced by H. uvarum. Currently, most studies on microbial volatiles are performed in vitro under nutrient rich conditions (Kai et al., 2009; Weise et al., 2012; Garbeva et al., 2014) and may not represent the conditions that prevail in the microbial environment. So in this work we studied the H. uvarum VOCs on strawberry. Furthermore, to study the antifungal activity of H. uvarum VOCs, we inoculated the B. cinerea on the wound strawberry for further research. All VOCs produced by microorganisms could generally be chemically grouped into alcohols, esters, alkanes, alkenes, alkynes, organic acids, ketones, terpenoids, aldehydes and sulfur

compounds (Corcuff et al., 2011; Wan et al., 2008). In this study, 28 volatiles were classified into alcohols, esters, organic acids, alkenes, ketones and aldehydes, which were in accordance with the above categories. We just presented the volatiles in every group (SI and RSI
800), and deducted the components of control. The rest VOCs were shown in Table 1. However, some researchers demonstrated that VOCs such as ethyl acetate; 3-methyl-1-butanol, acetate and hexanoic acid were detected in strawberries (Pelayo et al., 2003; Kim et al., 2013). Similarly, in our study, these volatiles were also detected in control (strawberries), but which were not in the scope (SI and RSI
800). Thus, these volatiles were not included in control when we analyzed the results. VOCs from strawberry inoculated B. cinerea alone were also detected in this work (data not shown), we found some volatiles such as 2-undecenal; 2-undecanone; 2decenal, (Z)-2-heptenal, (Z)-; 2H-pyran-2-one, tetrahydro-4hydroxy-4-methyl-; 2,4-decadienal; benzene, 4-ethenyl-1,2dimethyl- were not in other groups. Meanwhile, Ethyl acetate and 1,3,5,7-cyclooctatetraene were not detected in the strawberry inoculated with B. cinerea alone without H. uvarum (SI and RSI
800). The VOCs assay on strawberry was quite complex due to the interaction between strawberry and fungi. Some volatiles may be from yeast or strawberry induced by yeast and pathogen. In our previous work, volatiles from H. uvarum and B. cinerea on pure PDB cultures were detected. The results showed that H. uvarum VOCs including ethanol; ethyl acetate; propanoic acid, ethyl ester; 3methyl-1-butanol, 1-pentanol; 3-methyl-1-butanol, acetate;

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Fig. 3. Effect of the H. uvarum VOCs treatment on gray mold of strawberry. All groups were stored at 25  C after fumigation. Decay index and weight loss were determined on the 6th day. Bars represent standard errors of the mean of 3 independent replicates. Asterisks indicate statistical differences compared to control according to Duncan’s multiple range test at P  0.05 level.

Fig. 4. Effect of the volatiles of H. uvarum plus activated carbon on decay index and weight loss of strawberry stored at 25  C for 6 days. Bars represent standard errors of the mean of 3 independent replicates. Asterisks indicate statistical differences compared to control according to Duncan’s multiple range test at P  0.05 level.

1,3,5,7-cyclooctatetraene; phenylethyl alcohol; acetic acid; 2methyl-1propanol; acetic acid, 2-methylpropyl ester; acetic acid, pentyl ester; octanoic acid; octanoic acid, ethyl ester; propanoic acid, 2-methyl; hexanoic acid were detected. B. cinerea VOCs

including 2-methyl-1-butanol; butanoic acid, 2-methyl; 3-methyl1-butanol, acetate; hexanoic acid, methyl ester; benzaldehyde; benzene, 1,4-dichloro-; benzeneacetaldehyde; 1-undecene; 2nonanone; 2-nonanol were detected. According to the references, some volatiles in Table 1 have been demonstrated antifungal activity, such as ethanol; ethyl acetate; 3methyl-1-butanol; 1,3,5,7-cyclooctatetraene; phenylethyl alcohol; propanoic acid, ethyl ester; acetic acid, 2-phenylethyl ester; butanoic acid, ethyl ester; 2-nonanone; 3-methyl-1-butanol, acetate and phenol, 4-ethyl- (Table 1) (Huang et al., 2011; Singh et al., 2011; Kudalkar et al., 2012; Ren et al., 2010; Garbeva and De Boer, 2009; Garbeva et al., 2014; Chen et al., 2008; Druvefors et al., 2005; Stinson et al., 2003). Among these compounds, 1,3,5,7cyclooctatetraene, an antifungal compound (Huang et al., 2011; Stinson et al., 2003), which was not detected in strawberry, was enhanced by the presence of B. cinerea as relative peak area increased to 22.1%. This indicated that volatile compound was

Fig. 5. Effect of fumigation with H. uvarum VOCs on Decay index (A), Commodity rate (B), Weight loss (C), Firmness (D), pH (E) and TSS (F) of strawberry during cold storage. Bars represent standard errors of the mean of 3 independent replicates. Asterisks indicate statistical differences compared to control according to Duncan’s multiple range test at P  0.05 level.

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induced by B. cinerea. Some antifungal volatile compounds such as 2-nonanone, 3-methyl-1-butanol, acetate and phenol, 4-ethylwere detected in the presence of B. cinerea on strawberry. However, other volatiles detected in this study were needed to determine further for their antifungal activity by pure standard substance. Overall, We could combine with the previous work and indicate that H. uvarum produced some antifungal volatiles such as ethanol; propanoic acid, ethyl ester; 1,3,5,7-cyclooctatetraene and phenylethyl alcohol on pure culture and strawberries. How do the H. uvarum VOCs inhibit the mycelial growth and spore germination of B. cinerea? It is likely that different volatiles occur synergistically among the antagonist, pathogen and fruit. In vitro, B. cinerea grew the whole PDA plate almost on the 4th day and completely on the 5th day in the control. However, the mycelial growth and spore germination of B. cinerea were inhibited in the H. uvarum treatment. Furthermore, the antifungal effect of volatiles gradually increased with time (days) and the greatest inhibition (to 68.55%) of mycelial growth of B. cinerea was observed on the 3rd day (data not shown). Under optical light microscope, volatiles induced stunted tips and morphological abnormalities on the conidia of B. cinerea. This similar result was also found in other researches. For example, Using transmission electron microscopy, Li et al. (2012) revealed that fumigated and untreated B. cinerea showed excessive vesiculation or thickened cell walls in exposed conidia and increased vesiculation or strong retraction of plasma membrane in exposed hyphae. Bruce et al. (2003) showed that volatiles from bacteria and yeast inhibited pigment production by sapstain fungi. Thus, these results provided a better understanding of the volatiles’ mode of action. In order to verify the effect of H. uvarum VOCs on B. cinerea growth, spore suspensions (1  106 spore$mL1) were prepared from volatiles treatment and control on the 5th day and then inoculated in PDB. The results showed that there was no significant difference in spore production and germ tube length of B. cinerea between the control and the treatment in fresh PDB (data not shown). Thus, we speculated that H. uvarum only inhibited the mycelial growth and spore germination of B. cinerea via VOCs production, B. cinerea renewed growth when transferred to fresh PDB culture. In vivo, our results obtained that the infection process of B. cinerea on strawberry fruit was suppressed in the presence of volatiles. The volatiles mainly affected the early stages of the infection process by inhibiting conidial germination and mycelial growth, which were verified the results in vitro. Results of different fumigation time showed that the longer the time of fumigation, the greater the inhibitory activity against the B. cinerea. The reason maybe the longer fumigation time produced more antifungal volatile substances. Active carbon was mainly used to absorb gas. In order to verify the function of volatiles, we designed active carbon combined with H. uvarum as another control group. The results showed decay index of strawberry in additional active carbon and control group were significantly (P < 0.05) higher than that of H. uvarum treatment. However, the weight loss of strawberry in additional active carbon were significantly (P < 0.05) higher than that of other groups, including control. This phenomenon suggested that H. uvarum could inhibit significantly B. cinerea infection of strawberry via VOCs production. Meanwhile, we speculated that the PDA cultures may produce some volatiles which inhibit B. cinerea growth slightly. Some studies showed microbial VOCs display versatile functions: they inhibited bacterial and fungal growth, promote or inhibit plant growth, trigger plant resistance and attract other micro- and macro-organisms (Hagai et al., 2014; Schmidt et al., 2015). In the present study, the main quality parameters including firmness, total soluble solids, pH, weight loss, decay index

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and commodity of strawberry fruit were assayed. It was found that H. uvarum VOCs treatment maintained fruit weight loss, firmness, TSS and pH, and improved the commodity rate during cold storage. This also indicated that H. uvarum VOCs not only inhibited the B. cinerea growth, but decreased the fruit rot and prolonged the storage time. In conclusion, the current work demonstrated that the volatiles from H. uvarum could inhibit B. cinerea in vitro and on strawberry fruit, and could potentially be an effective alternative for the control of postharvest diseases by fumigant action. Further studies are needed on improving the production of VOCs from H. uvarum and testing the inhibitory effect on other pathogens. Funding This study was supported by Science and Technology Department of Jiangsu Province (No. BE2010385) and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions and National College Students’ innovation and entrepreneurship (No. 201310307046). Notes The authors declare no competing financial interest. References Basha, H., Ramanujam, B., 2015. Growth promotion effect of Pichia guilliermondii in chilli and biocontrol potential of H. anseniaspora uvarum against Colletotrichum capsici causing fruit rot. Biocontrol Sci. Technol. 25 (2), 185e206. Bruce, A., Stewart, D., Verrall, S., Wheatley, R.E., 2003. Effect of volatiles from bacteria and yeasts on the growth and pigmentation of sapstain fungi. Int. Biodeter. Biodegrad. 51, 101e108. Cai, Z.K., Yang, R., Xiao, H.M., Qin, X.J., Si, L.Y., 2015. Effect of preharvest application of Hanseniaspora uvarum on postharvest diseases in strawberries. Postharvest Biol. Tec. 100, 52e58. Chen, H., Xiao, X., Wang, J., Wu, L.J., Zheng, Z.M., Yu, Z.L., 2008. Antagonistic effects of volatiles generated by Bacillus subtilis spore germination and hyphal growth of the plant pathogen, Botrytis cinerea. Biotechnol. Lett. 30, 919e923. Corcuff, R., Mercier, J., Tweddel, R., 2011. Effect of water activity on the production of volatile organic compounds by Muscodor albus and their effect on three pathogens in stored potato. Fungal Biol. 115, 220e227. Costa, L.B., Rangel, D.E.N., Morandi, M.A.B., Bettiol, W., 2013. Effects of UV-B radiation on the antagonistic ability of Clonostachys rosea to Botrytis cinerea on strawberry leaves. Biol. Control 65, 95e100. Dennis, C., 1978. Post-harvest spoilage of strawberries. ARC Res. Rev. 4, 38e44. Di Francesco, A., Ugolini, L., Lazzeri, L., Mari, M., 2015. Production of volatile organic compounds by Aurebasidium pullulans as a potential mechanism of action against postharvest fruit pathogens. Biol. Control 81, 8e14. Droby, S., Lichter, A., 2004. Post-harvest Botrytis infection: etiology, development and management. In: Botrytis: Biology, Pathology and Control, pp. 349e367. Druvefors, A.U., Passoth, V., Schnurer, J., 2005. Nutrients effects on biocontrol of Penicillium roqueforti by Pichia anomala J121 during airtight storage of wheat. Appl. Environ. Microbiol. 71, 1865e1869. El-Neshawy, S.M., Shetaia, Y.M.H., 2003. Biocontrol capability of Candida spp. Against Botrytis rot of strawberries with respect to fruit quality. In: Tijskens, L.M.M., Vollebregt, H.M. (Eds.), Proceedings of the International Conference on Quality in Chains, Vols. 1 and vol. 2. An Integrated View on Fruit and Vegetable Quality. 727e733. Fan, Y., Xu, Y., Wang, D., Zhang, L., Sun, J., Sun, L., Zhang, B., 2009. Effect of alginate coating combined with yeast antagonist on strawberry (Fragaria  ananassa) preservation quality. Postharvest Biol. Technol. 53, 84e90. Garbeva, P., De Boer, W., 2009. Inter-spectic internations between carbon-limited soil bacteria affect behavior and gene expression. Microb. Ecol. 58, 36e46. Garbeva, P., Hordijk, C., Gerards, S., De Boer, W., 2014. Volatiles produced by the mycophagous soil bacterium Collimonas. FEMS Microbiol. Ecol. 87, 639e649. Hagai, E., Dvora, R., Havkin-blank, T., Zelinger, E., Porat, Z., Schulz, S., Helman, Y., 2014. Surface-motility induction, attraction and hitchhiking between bacterial species promote dispersal on solid surfaces. ISME J. 8, 1147e1151. Huang, R., Li, G.Q., Zhang, J., Yang, L., Chen, H.J., Jiang, D.H., Hunag, H.C., 2011. Control of postharvest Botrytis fruit rot of strawberry by volatile organic compounds of Candida intermedia. Phytopathology 101, 859e869. Kai, M., Haustein, M., Molina, F., Petri, A., Scholz, B., Piechulla, B., 2009. Bacterial volatiles and their action potential. Appl. Microbiol. Biotechnol. 81, 1001e1012. Kim, Y.H., Kim, K.H., Szulejko, J.E., Parker, D., 2013. Quantitative analysis of fragrance and odorants released from fresh and decaying strawberries. Sensors 13 (6),

212

X. Qin et al. / Food Microbiology 63 (2017) 205e212

7939e7978. Kudalkar, P., Strobel, G., Riyaz-UI-Hassan, S., Geary, B., Sears, J., 2012. Muscodor sutura, a novel endophytic fungus with volatile antibiotic activities. Mycoscience 53, 319e325. Li, Q.L., Ning, P., Zheng, L., Huang, J.B., Li, G.Q., Hsiang, T., 2010. Fumigant activity of volatiles of Streptomyces globisporus JK-1 against Penicillium italicum on Citrus microcarpa. Postharvest Biol. Tec. 58, 157e165. Li, Q.L., Ning, P., Zheng, L., Huang, J.B., Li, G.Q., Hsiang, T., 2012. Effects of volatile substances of Streptomyces globisporus JK-1 on control of Botrytis cinerea on tomato fruit. Biol. Control 61, 113e120. Li, W.H., Zhang, H.Y., Li, P.X., Apaliya, M.T., Yang, Q.Y., Peng, Y.P., Zhang, X.Y., 2016. Biocontrol of postharvest green mold of oranges by Hanseniaspora uvarum Y3 in combination with phosphatidylcholine. Biol. Control 103, 30e38. Liu, H.M., Guo, J.H., Luo, L., Liu, P., Wang, B.Q., Cheng, Y.J., Deng, B.X., Long, C.A., 2010. Improvement of Hansenisapora uvarum biocontrol activity against gray mold by the addition of ammonium molybdate and the possible mechanisms involved. Crop Prot. 29, 277e282. Maryam, Z.Z., Sabihe, S.Z., Mahmoud, S.Z., Sayed, A.H.G., 2014. Integration of Lactobacillus plantarum A7 with thyme and cumin essential oils as a potential biocontrol tool for gray mold rot on strawberry fruit. Postharvest Biol. Tec. 92, 149e156. Mercier, J., Smilanick, J.L., 2005. Control of green mold and sour rot of stored lemon by biofumigation with Muscodor albus. Biol. Control 32, 401e407. Moreira, N., Pina, C., Mendes, F., Couto, J.A., Hogg, T., Vasconcelos, I., 2011. Volatile compounds contribution of Hanseniaspora guilliermondii and Hanseniaspora uvarum during red wine vinifications. Food Control 22, 662e667. Parafati, L., Vitale, A., Restuccia, C., Cirvilleri, G., 2015. Biocontrol ability and action mechanism of food-isolated yeast strains against Botrytis cinerea causing postharvest bunch rot of table grape. Food Microbiol. 47, 85e92. Pelayo, C., Ebeler, S.E., Kader, A.A., 2003. Postharvest life and flavor quality of three strawberry cultivars kept at 5  C in air or airþ20 kPa CO2. Postharvest Biol. Tec. 27 (2), 171e183. Platania, C., Restuccia, C., Muccilli, S., Cirvilleri, G., 2012. Efficacy of killer yeasts in the biological control of Penicillium digitatum on Tarocco orange fruits (Citrus sinensis). Food Microbiol. 30, 219e225. Prusky, D., Gullino, M.L., 2010. Post-harvest Pathology. Springer, Dordrecht. Qin, X.J., Xiao, H.M., Xue, C.H., Yu, Z.F., Yang, R., Cai, Z.K., 2015. Biocontrol of gray mold in grapes with the yeast Hanseniaspora uvarum alone and in combination with salicylic acid or sodium bicarbonate. Postharvest Biol. Tec. 100, 160e167. Ren, Y.H., Strobel, G., Sears, J., Park, M., 2010. Geobacillus sp., a thermophilic soil

bacterium producing volatile antibiotics. Microb. Ecol. 60, 130e136. Romanazzi, G., Nigro, F., Ippolito, A., Salerno, M., 2001. Effect of short hypobaric treatments on postharvest rots of sweet cherries, strawberries and table grapes. Postharvest Biol. Tec. 22, 1e6. Romanazzi, G., Lichter, A., Gabler, F.M., Smilanick, J.L., 2012. Recent advances on the use of natural and safe alternatives to conventional methods to control postharvest gray mold of table grapes. Postharvest Biol. Tec. 63, 141e147. Rouissi, W., Ugolini, L., Martini, C., Lazzeri, L., Mari, M., 2013. Control of postharvest fungal pathogens by antifungal compounds from Penicillium expansum. J. Food Prot. 11, 1879e1886. Schmidt, R., Cordovez, V., De Boer, W., Raaijmakers, J., Garbeva, P., 2015. Volatile affairs in microbial interactions. ISME J. 42, 1e7. Singh, S.K., Strobel, G.A., Knighton, B., Geary, B., Sears, J., Ezra, D., 2011. An Endophytic Phomopsis sp. possessing bioactivity and fuel potential with its volatile organic compounds. Microb. Ecol. 61, 729e739. Spadaro, D., Droby, S., 2016. Development of biocontrol products for postharvest diseases of fruit: the importance of elucidating the mechanisms of action of yeast antagonists. Trends Food Sci. Tech. 47, 39e49. Stinson, M., Ezra, D., Hess, W.M., Sears, J., Strobel, G., 2003. An endophytic Gliocladium sp. Of Eucryphia cordifolia producing selective volatile antimicrobial compounds. Plant Sci. 165, 913e922. Wan, M., Li, G., Zhang, J., Jiang, D., Huang, H.C., 2008. Effect of volatile substances of Streptomyces platensis F-1 on control of plant fungal disease. Biol. Control 46, 552e559. Wei, Y.Y., Mao, S.B., Tu, K., 2014. Effect of preharvest spraying Cryptococcus laurentii on postharvest decay and quality of strawberry. Biol. Control 73, 68e74. Weise, T., Kai, M., Gummesson, A., Troeger, A., von Reuss, S., Piepenborn, S., Kosterka, F., Sklorz, M., Zimmermann, R., Francke, W., Piechulla, B., 2012. Volatile organic compounds produced by the phytopathogenic bacterium Xanthomonas campestris pv. vesicatoria 85-10. Beilstein J. Org. Chem. 8, 579e596. Zhang, H., Wang, L., Dong, Y., Jiang, S., Cao, H., Meng, R., 2007. Postharvest biological control of gray mold decay of strawberry with Rhodotorula glutinis. Biol. Control 40, 287e292. Zhang, H.Y., Ge, L.L., Chen, K.P., Zhao, L.N., Zhang, X.Y., 2014. Enhanced biocontrol activity of Rhodotorula mucilaginosa cultured in media containing chitosan against postharvest diseases in strawberries: possible mechanisms underlying the effect. J. Agric. Food Chem. 62, 4214e4224. Zheng, M., Shi, J.Y., Shi, J., Wang, Q.G., Li, Y.H., 2013. Antimicrobial effects of volatiles produced by two antagonistic Bacillus strains on the anthracnose pathogen in postharvest mangos. Biol. Control 65, 200e206.